www.nature.com/scientificreports OPEN Ancient human mitochondrial genomes from Bronze Age Bulgaria: new insights into the Received: 26 July 2018 Accepted: 20 March 2019 genetic history of Thracians Published: xx xx xxxx Alessandra Modi1, Desislava Nesheva2, Stefania Sarno3, Stefania Vai 1, Sena Karachanak- Yankova2, Donata Luiselli4, Elena Pilli1, Martina Lari1, Chiara Vergata1, Yordan Yordanov5, Diana Dimitrova6, Petar Kalcev7, Rada Staneva2, Olga Antonova2, Savina Hadjidekova2, Angel Galabov8, Draga Toncheva2 & David Caramelli1 One of the best documented Indo-European civilizations that inhabited Bulgaria is the Thracians, who lasted for more than fve millennia and whose origin and relationships with other past and present- day populations are debated among researchers. Here we report 25 new complete mitochondrial genomes of ancient individuals coming from three necropolises located in diferent regions of Bulgaria – Shekerdja mogila, Gabrova mogila and Bereketska mogila – dated to II-III millennium BC. The identifed mtDNA haplogroup composition refects the mitochondrial variability of Western Eurasia. In particular, within the ancient Eurasian genetic landscape, Thracians locate in an intermediate position between Early Neolithic farmers and Late Neolithic-Bronze Age steppe pastoralists, supporting the scenario that the Balkan region has been a link between Eastern Europe and the Mediterranean since the prehistoric time. Spatial Principal Component Analysis (sPCA) performed on Thracian and modern mtDNA sequences, confrms the pattern highlighted on ancient populations, overall indicating that the maternal gene pool of Thracians refects their central geographical position at the gateway of Europe. Bulgaria is situated in the eastern part of the Balkan Peninsula, at the connection point between Southeastern Europe, Eurasian steppe, Anatolia and the Aegean islands. Te presence of modern humans in this region is attested starting from 40 kya by the Paleolithic series at Bacho Kiro and Temnata Dupka Caves1–3. Some archaeo- logical sites associated with early farmers, as well as the earliest evidence of copper metallurgy in Europe, indicates that this area played a signifcant role both in the Neolithic and in the Metal Ages4. One of the best documented Indo-European civilizations that inhabited Bulgaria consists in the Tracians, whose cultural legacy is still evident in the modern society. Diferent theories have been historically proposed about the origin of the Tracians. Today it is assumed that the Tracian culture emerged and formed in the early Bronze Age5–7, a period characterized by strong cul- tural changes and movements of people westward from the Steppe8. During the 5th and 4th millennium BCE, the inhabitants of the eastern region of Balkans were organized in diferent groups of indigenous people that, over time, were named under the single ethnonym of “Tracians”9–11. According to historical and archaeological sources, the Tracian culture fourished during the 2nd and 3rd millennia BCE12,13. Te rich cultural and histori- cal heritage, represented by fortresses and necropolises, as well as by the world-famous Panagyurishte, Valchitran, Lukovit and Rogozen treasures, dates back to this period. In the later periods, several populations (Greeks, 1Department of Biology, University of Florence, Florence, Italy. 2Department of Medical Genetics, Medical University of Sofa, Sofa, Bulgaria. 3Department of Biological, Geological and Environmental Sciences, University of Bologna, Bologna, Italy. 4Department of Cultural Heritage, University of Bologna, Ravenna, Italy. 5Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofa, Bulgaria. 6The Regional Historical Museum of Sliven, Sliven, Bulgaria. 7The Regional Historical Museum of Stara Zagora, Stara Zagora, Bulgaria. 8The Stephan Angelof Institute of Microbiology, Bulgarian Academy of Sciences, Sofa, Bulgaria. Alessandra Modi, Desislava Nesheva and Stefania Sarno contributed equally. Correspondence and requests for materials should be addressed to D.T. (email: [email protected]) or D.C. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:5412 | https://doi.org/10.1038/s41598-019-41945-0 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. Geographical location of the necropolises in Bulgaria. Black triangles specifcally indicate the locations of the considered archaeological sites while the grey dots refer to current Bulgarian provinces. Te map is plotted using https://www.freepik.com/, processed with Adobe Illustrator CS6 and modifed with Photoshop CS6 (2012) by Dimitar Spassov - web developer ([email protected]) and Desislava Nesheva. Image is attributed to valeria_aksakova/Freepik. Macedonians, Slavs and proto-Bulgarians) arrived in the Balkans, reaching the lands occupied by Tracians and mixing with them, thus infuencing their cultural and biological identity11. Genetic analyses on both autosomal variations14 and uniparental genetic markers15–17 of present-day Bulgarians, locate them between Eastern European and Mediterranean populations, with a particular afnity to the neighboring groups from Greece and the Balkans16. In addition, the Bulgarian maternal genetic pool particu- larly suggests a major Western Eurasian origin, tracing their ancestry to lineages that witness a complex genetic structure of the region today and refect diferent peopling and admixture events from the Upper Paleolithic to the onset of the Neolithic and Post-Neolithic in Europe15. Recent genome-wide ancient DNA (aDNA) based stud- ies on Southeastern Europe, have shown that Neolithic population from present-day Bulgaria was closely related with the northwestern-Anatolian-Neolithic ancestry that signals the spread of Early farmers across Europe, except for the individuals lived in the mid-sixth millennium BC in Malak Preslavets, who revealed a signifcantly higher level of hunter-gatherer-related ancestry than the other Balkan Neolithic individuals18. Starting from the early 3rd millennium BCE, migrations from the adjacent Pontic-Caspian and Eurasian steppe also played an important role in the transformation of the European genetic landscape, and the contribution of Steppe ancestry to Southeastern European populations increased particularly during the Bronze Age18. Although our under- standing of the population and cultural dynamics occurred in the (pre-) history of Balkan Peninsula is starting to be increasingly elucidated, the genetic details on the local civilization remain unknown and this information is only partly available for the ancient (proto-) Bulgarian people18,19. We now have the opportunity to investigate the genetic structure of the Tracians, an ancient people that lasted for more than fve millennia and whose origin and relationships with other past and present-day populations are still debated among researchers. To investigate the genetic structure and population history of this ancient civilization, we analyzed 25 com- plete mitochondrial genomes from three Tracian necropolises (Fig. 1 and Table 1) along with modern and ancient European data. Te characterization of the Tracian mitochondrial DNA (mtDNA) variability may have important implications for understanding the dynamics of interaction between Eastern Europe and the Mediterranean, and will also contribute to better clarify the genetic evolution of European populations and the origin of contemporary Bulgarian gene pool. Results We successfully reconstructed complete or almost entire mitochondrial genomes for 26 individuals, 3 from Shekerdja mogila, 1 from Gabrova mogila and 22 from Bereketska mogila (Table 2). All the resulted sequences reach the standard quality requested to guaranty the reliability of the NGS data; CtoT patterns range between 20% to 46%, average fragment size vary from 44.4 base pair (bp) to 67.4 bp and no signifcant levels of present-day human contamination were detected (Table 2). Only one sample, BM-51, showed a high level of contamination and was not considered in the following statistical analyses. Te direct radiocarbon dating performed on the samples BM 44, SM 8.1 and GM 30.3 placed the remains at II-III millennium BC (CEDAD, Centro di DAtazione e Diagnostica, Univerità del Salento, Italy) (Table 1), that corresponds to the age estimated according to the archaeological record. Te mtDNA sequences obtained were assigned to 21 diferent haplogroups, representative of the mitochondrial variability of Western Eurasia (Table 2 and Supplementary Table S1). Phylogenetic links between haplotypes of the Tracian samples and comparison ancient data are shown in the Median Joining Network (Fig. 2). Most of the Tracian individu- als belong to sub-lineages of the macro-haplogroup H, which accounts for an overall frequency of 33%. Tis is the most frequent mitochondrial lineage in present-day Europe, representing over 40% of the total mtDNA variability20. Its frequency observed in the Tracians samples is almost similar to the frequency in contempo- rary European population. Two individuals belong to haplogroup HV, an ancient European lineage likely orig- inating in the Mediterranean region during the Last Glacial Maximum (LGM)21. In ancient samples, HV has been identifed in one Mesolithic specimen from Sicily22 and in early Neolithic remains from Spain23, Germany8 and Russia18,24; Mathieson et al.18 reported a HV haplotype in one sample from Serbia dating from 5800 BCE. SCIENTIFIC REPORTS | (2019) 9:5412 | https://doi.org/10.1038/s41598-019-41945-0